基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化、纯化和检测方法  

A Method for Biotinylation,Purification and Detection of Enhanced Green Fluorescent Protein Based on Avi-tag Technology in Human Embryonic Kidney Cells

作　　者：李丹丹[1,2] 李英[2,3] 吴小桃[2] 鲁云霞[1] 王学军[2] 王升启[2] 

机构地区：[1]安徽医科大学,安徽合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]天津中医药大学,天津300193

出　　处：《生物技术通讯》2011年第5期647-650,666,共5页Letters in Biotechnology

基　　金：国家传染病防治科技重大专项(2008zx10002-011);国家杰出青年基金(30625041)

摘　　要：目的:建立并优化基于Avi-tag标签技术的人胚肾细胞增强型绿色荧光蛋白(eGFP)的定点生物素化标记、纯化和检测方法。方法:分别构建具有Avi-tag标签的eGFP真核表达载体plenti-Avi-eGFP和BirA酶真核过表达载体pQCXIH-BirA,将plenti-Avi-eGFP和pQCXIH-BirA共转染人胚肾293T细胞,12 h后观察Avi-tag标签对eGFP蛋白在细胞内定位的影响;48 h后裂解细胞,用链霉亲和素珠子纯化生物素标记的eGFP,SDS-PAGE观察eGFP纯化和富集情况,并优化基于Western印迹的生物素化eGFP检测方法。结果:Avi-tag标签对eGFP在细胞内的定位无影响,同时BirA酶在293T细胞内可将带Avi-tag标签的eGFP标记上生物素;生物素化的eGFP可特异性地被链霉亲和素珠子纯化和富集,纯度可达95%;Western印迹检测生物素化蛋白的最终条件为5%的BSA作为封闭液和终浓度为100 ng/mL的链霉亲和素-HRP。结论:建立了基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化标记、纯化与检测方法,为该方法的广泛应用奠定了前期技术基础。Objective： To establish and optimize a method for site-specific biotinylation,purification and detection of enhanced green fluorescent protein（eGFP） in human embryonic kidney cells based on Avi-tag technology.Methods： We respectively construct the Avi-tagged eGFP eukaryotic expression vector plenti-Avi-eGFP and BirA enzyme over-expression eukaryotitc vector pQCXIH-BirA,then plenti-Avi-eGFP and pQCXIH-BirA were co-trans-fected into human embryonic kidney cells 293T.After 12 h,we observed that whether the Avi-tag have an impact on the location of eGFP in the cells.After 48 h the 293T cells were cracked and biotinylated eGFP was purified from lysis supernatant by using streptavidin beads.SDS-PAGE was used to observe the situation of purification and enrichment and then the condition of detecting biotinylated eGFP based on Western blotting was optimized.Re-sults： The Avi-tag had no effect on the location of eGFP in the cells.At the same time the Avi-tagged eGFP was successfully biotinylated by BirA in 293T cells,and the biotinylated eGFP was purified and enriched specifically by streptavidin beads,and the purity of eGFP reached to 95%.The ultimate terms for detecting biotinylated protein based on Western blotting were determinated as 5% BSA as the blocking reagent and 100 ng / mL streptavidin-HRP as working solution.Conclusion： The Avi-tag method for site-specific biotinylation,purification and detection of the eGFP in human embryonic kidney cells is set up in our lab.

关 键 词：Avi-tag 增强型绿色荧光蛋白 BirA酶 人胚肾细胞 亲和纯化 

分 类 号：Q503[生物学—生物化学] Q78